G protein-coupled receptors (GPCRs) mediate hormonal control of numerous signaling pathways. Many of these pathways are dynamically regulated. At the level of the receptor, regulation can occur via inhibition of GPCR/G protein coupling (desensitization), redistribution of cell surface receptors (trafficking), or receptor degradation (down-- regulation). Two protein families, G protein-coupled receptor kinases (GRKs) and arrestins, play a critical role in regulating these processes. GRKs specifically phosphorylate the activated form of the receptor, which in turn promotes arrestin binding. Arrestin interaction has been directly linked to many regulatory processes including GPCR desensitization and trafficking. In addition, recent studies suggest a role for arrestins in GPCR signaling via non-receptor tyrosine kinase and MAP kinase pathways. In the previous grant period we focused on characterizing the role of arrestins in regulating GPCR desensitization and trafficking in intact cell models. In the present application we propose to continue our analysis of arrestin function by addressing three major questions. 1. Do GPCRs contain discrete arrestin-binding motifs that regulate the specificity of arrestin/receptor interaction? While arrestins bind to GPCRs in an activation and phosphorylation-dependent manner, little is known about specific receptor motifs that enable arrestins to bind in an activation-dependent manner. We will attempt to identify and characterize such motifs using both biochemical and cell-based strategies. 2. What are the critical interactions that mediate arrestin regulation of GPCR endocytosis and recycling? The ability of arrestins to mediate GPCR endocytosis appears to be regulated via interactions with clathrin, beta-adaptin, and PIP2. We will further probe these interactions in an effort to clarify the mechanistic basis for arrestin-- mediated regulation of GPCR endocytosis. In addition, since recent studies suggest that arrestins also regulate the rate of GPCR recycling, we will test the hypothesis that this is mediated via arrestin interaction with proteins directly involved in vesicular trafficking. 3. Do GPCRs regulate additional interactions that contribute to the diverse nature of arrestin function? This will be addressed using both biochemical and cell-based strategies to search for differential binding partners of wild type and constitutively active arrestins. Candidate interacting proteins will be isolated, identified, and characterized by analyzing their effect on arrestin-mediated desensitization, trafficking and signaling of selected GPCRs. Overall, our efforts should provide unique mechanistic insight into the biochemical, cellular and molecular function of arrestins.